The long term goal of this project is to understand how ribosomes are assembled in eukaryotes. We use the yeast Saccharomyces cerevisiae, to facilitate combined genetic, molecular biological, biochemical, and proteomic approaches. Ribosome assembly initiates in the nucleolus, where rRNA is transcribed, associates with ribosomal proteins, and undergoes modification and initial processing to begin to form mature ribosomal RNPs. Subsequent steps in maturation of preribosomal particles occur upon their release from the nucleolus to the nucleoplasm and upon their export to the cytoplasm. This assembly pathway requires a dynamic series of remodeling steps in which protein-protein, RNA-protein, and RNA-RNA interactions are established, disrupted and reconfigured. Dysregulation of this pathway in humans leads to many diseases related to alterations in cell growth or proliferation, including cancer. Screens for yeast mutants defective in ribosome biogenesis, and development of methods to purify ribosome assembly intermediates and identify their constituents, led to the identification of ~180 trans-acting factors required for ribosome assembly. Central to understand the mechanisms of ribosome biogenesis will be to figure out the precise roles played by each of these assembly factors. Which proteins interact with each other? Which proteins contact RNA? Can one define assembly neighborhoods within the nascent rRNPs, as observed for prokaryotic ribosomes assembled in vitro? In what order do these factors associate with preribosomes, carry out their functions, then dissociate from the particles? By what means are assembly factors and ribosomal proteins recruited to pre-ribosomes, activated, then released from the pre-rRNPs? Experiments are proposed to address these questions. We are focusing on two particular consecutive steps in the maturation of precursors to mature 60S ribosomal subunits--maturation of the 66SA3 assembly intermediates, followed by the 66SB particles. To begin to establish paradigms for mechanisms of factor function, we will address the above questions for several factors required for these two steps in assembly. In addition, we will investigate in more detail the role of the DEAD-box protein Drs1. Does this putative ATPase enable maturation of 66SB particles, by triggering the release of negative regulators that bind to pre-rRNA and prevent its premature cleavage? The high degree of conservation of molecules involved in eukaryotic ribosome biogenesis promises that principles governing ribosome biogenesis discovered in yeast will provide blueprints to study more diverse modes of regulation of ribosome assembly in metazoans, including those disrupted in many diseases. PUBLIC HEALTH RELEVANCE Ribosomes are ribonucleoprotein particles present in almost every cell of every organism that catalyze protein synthesis by translation of the genetic code in messenger RNA. Thus these machines are essential for and closely related to the growth, proliferation, and adaptation of cells. The goal of our research is to understand how the protein and RNA constituents of ribosomes assemble together to make functional protein synthesis machines. The nucleolus, the subcompartment of the cell where ribosomes are assembled, is also home to many other molecules that regulate important cellular events, for example, stem cell differentiation, neurological function, and aging. Mutations in ribosome assembly factors or ribosomal proteins perturb ribosome assembly and nucleolar morphology, and lead to diseases such as cancer. The frequent connection between aberrant nucleolar morphology and disease has lead to using nucleolar morphology as a diagnostic marker for malignancies.